1. Field of the Invention
Embodiments of the invention relate to treatment of a viral infection with a monoclonal antibody to thymidine kinase.
2. Description of the Related Art
Thymidine kinase (ATP:thymidine-5′ phosphotransferase; EC 2.7.1.21 in the International Union of Biochemistry classification system) is an enzyme that phosphorylates thymidine to thymidine monophosphate (TMP). The commonly used abbreviation of TK will be used herein to denote thymidine kinase in a general sense, where different TK isozymes are not specified particularly.
Thymidine kinase protein has been isolated from many different sources and purified to varying degrees. A variety of different molecular weight thymidine kinases have been reported from human samples, depending on the particular cell and the method of isolation and analysis. In general, the findings suggest that thymidine kinase exists in at least one monomeric form and a variety of multimeric forms.
In humans, it is also known that there are at least two major isozymes (similar but distinct forms) of thymidine kinase, referred to herein as TK1 and TK2. These isozymes are produced from different genes, are found in different cellular compartments, and differ in their levels and timing of expression during the cell cycle and according to the cell differentiation state. In humans, the TK1 gene is on chromosome 17 in band q21-22 (Elsevier 1974) while the TK2 gene is on chromosome 16 (Willecke et al. (1977) Somatic Cell Genet. 3:237). A gene for TK1 has been cloned and sequenced (Lin (1984) Proc. Nat'l Acad. Sci. 81:414-418; Flemington (1987) Gene 52:267-277).
There are extensive inconsistent reports in the prior art on the properties of mammalian TK1, with diverging results and observations as to the electrophoretic behavior and kinetic properties. Native molecular weights between 45,000 and 200,000 daltons have been reported for the native human TK1 from, for example, leukemic cells (96 kD, Sherley et al. (1988) J. Biol. Chem 263:375-391; 150-200 kD, Munch-Petersen et al., (1990) Leuk. Res. 14:39-45), human placenta (45 kD, Ellims et al. (1982) Mol. Cell. Biochem. 45:113-116); 92 kD, Gan et al. (1983) J. Biol. Chem. 258:7000-7004; 70 kD, Tamiya et al. (1989) Biochim. Biophys. Acta 995:28-35), lymphocytes (110 kD, Munch-Petersen et al. (1991) J. Biol. Chem. 266:9032-9038), and human breast cancer (177 kD, Bronzert et al. (1981) Cancer Res. 41:604-610).
It has been reported that in the presence of ATP, native TK1 shifts to a form of TK1 having a higher molecular weight, for example, human placental TK1 of 50 kD shifts to 70 kD in the presence of ATP (Tamiya et al. (1989), supra) and human lymphocytic TK1 of 55 kD shifts in the presence of ATP to a form having a molecular weight of 110 kD (Munch-Petersen et al. (1991) supra).
Not only are widely divergent values reported for the molecular weight of the native TK1, different views exist in the prior art for the monomeric subunit of TK1. Molecular weights of 44 and 22-24 kD have been reported for the TK1 monomer. Further, reports vary as to whether the monomeric subunit is associated with TK1 enzymatic activity. For example, TK1 enzyme activity has been reported to be associated with the monomeric subunit of approximately 24 kD for the HeLa cells (Sherley et al. (1988) supra), rat liver (Baron et al. (1990) Preparative Biochemistry 20:241-256), and human lymphocytes (Munch-Petersen (1991) supra), but enzyme activity was not found associated with the monomeric subunit in the presence or absence of ATP for human placenta TK1 (Tamiya et al. (1989) supra).
Balis et al. (U.S. Pat. No. 4,317,877, Mar. 2, 1982) disclosed immunesera to a small subunit component of (a) TK from normal colonic mucosa and (b) TK from term human placenta. Although both small subunit components were electrophoretically similar, they were not antigenically identical as indicated by differences in precipitin patterns. Moreover, it was stated that “The lack of complete neutralization by these antisera of their respective homologous enzymes is not unexpected since only the small molecular weight component is used as antigen.” The teaching in the Balis et al. patent, supra, is that an antiserum to a subunit component of TK1 does not completely react with nor neutralize the active multimeric form of the TK1. Also, the Balis antibody did not react with leukemic leukocytes or with normal or mitogen-stimulated peripheral lymphocytes, even though these are known to have elevated TK levels (Balis et al., col. 2, lines 21-23).
Another European Patent publication, No. 0 255 431 by Jouan published Oct. 23, 1991, discloses purification of “TK-F” (fetal TK or TK1) from human placental material for purposes including the use of the pure TK-F to produce anti-TK-F antibodies. Jouan teaches the purification of TK-F using prior art technology which has been shown in various reports to result in the purification of a TK1 so labile that yields of purified TK are insufficient for further manipulation, e.g., for biochemical characterization, monoclonal antibody preparation, screening, etc. Jouan suggests the use of art-known methods to prepare monoclonal antibody using his purified TK-F, however, the patent does not teach how to overcome the problem of extreme lability associated with a purified TK1 obtained using prior art methodologies, a problem noted in many prior art references.
U.S. Pat. No. 5,698,409, issued Dec. 16, 1997, which is incorporated herein by reference, describes a purified mammalian thymidine kinase 1 (TK1) from Raji cells and a TK1 monoclonal antibody. Raji cells are an immortalized human lymphoma cell line, available from ATCC as cell line #CCL-86. U.S. Pat. No. 5,698,409 also describes a monoclonal antibody to TK1 which not only binds to TK1 but also inhibits TK1 activity. Specific anti-TK1 antibody monoclonal producing hybridomas are available as ATCC HB 11432, HB 11433, HB 11434, and PTA-6704.
TK-1 is a cellular enzyme which is involved in a “salvage pathway” of DNA synthesis. In normal growing cells thymidine kinase 1 mRNA rises near the G1-S boundary, peaks in early S phase, and returns in G2 to approximately the level of early G1. It is activated in the G1/S phase of the cell cycle, and its activity has been shown to correlate with the proliferative activity of tumor cells. Proliferating cells appear to have lost the strict regulation of TK1 that is observed in normal cells. TK activity is a major biochemical marker of cell proliferation and several studies show that TK levels are elevated in malignancies.
In DNA tumor virus-transformed cells, the level of TK mRNA remains relatively constant throughout all phases of the cell cycle. Data suggest that DNA tumor viruses suppress a transcriptional down-regulation common to enzymes responsible for the DNA precursor pathway. In virally transformed cells lines both TK1 mRNA levels and TK1activity remain elevated throughout the cell cycle (Different regulation of thymidine kinase during the cell cycle of normal versus DNA tumor virus-transformed cells. Hengstschlager, M., Knofler, m., Mullner, E. W., Ogris, E., Wintersberger, E., Wawra, E. J. Biol. Chem., 269: 13836-13842, 1994). The step catalysed by thymidine kinase 1 is the bottle neck of the S-phase gene pathway and is therefore rate limiting. Even slow-growing cancers or latent viral infections constitutively express TK1 on the cell surface making them susceptible to ADCC and CDC (A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation. Hengstschlager M, Mudrak I, Wintersberger E, Wawra E. Cell Growth Differ. 1994 December;5(12):1389-94. Vienna Biocenter, University of Vienna, Austria).
Relationship to TK1 and HIV, CMV, HCV, Papilloma, Polyoma, Adenovirus, etc . . .
T cell synthesis of dNTP by Thymidine Kinase is required by HIV for reverse transcription by reverse transcriptase and integration of the viral genome into the host DNA. The site of viral genome integration of TK1 in SHIV infection results in the over-expression of thymidine kinase mRNA, which is abolished by highly active antiretroviral therapy (HAART) The expression of P-glycoprotein and cellular kinases is modulated at the transcriptional level by infection and highly active antiretroviral therapy in a primate model of AIDS (Jorajuria S, Clayette P, Dereuddre-Bosquet N, Benlhassan-Chahour K, Thiebot H, Vaslin B, Le Grand R, Dormont D. AIDS Res Hum Retroviruses. 2003 April;19(4):307-11. CEA, Service de Neurovirologie, DRM/DSV, CRSSA, EPHE, IPSC, 92265 Fontenay-aux-Roses, France).
Likewise Cytomegaloviruses (CMVs) do not encode many of the biosynthetic enzymes for DNA precursor synthesis. The virus requires a mechanism to overcome cellular quiescence. HCMV infection induces the progression of quiescent cells toward the G1_S transition point and activates the TK1 gene required for DNA replication. When HFF cells were infected with HCMV Towne strain, and cytoplasmic RNAs were harvested at 6 and 24 h after infection, there was an increase in steady-state RNA levels of many genes for cell cycle progression to the G1_S transition point (Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: A DNA microarray analysis. Yoon-Jae Song and Mark F. Stinski. PNAS Mar. 5, 2002, vol. 99, no. 5). All of these studies indicate that levels of TK1 are elevated during periods of cell proliferation such as viral infection.
Likewise, in cells transformed with polyoma virus, papilloma virus, adenovirus, or SV40, TK activity as well as TK mRNA was consistently higher in all phases of the cell cycle, and TK mRNA never displayed significant cell cycle-dependent changes. Furthermore, it is possible to up-regulate TK mRNA and enzyme activity throughout the normal cell cycle simply by expressing the polyoma large T antigen in a cell line carrying the information for this protein in inducible form. This work presents evidence that DNA tumor viruses have a mechanism to keep TK expression high during the cell cycle (“Different regulation of thymidine kinase during the cell cycle of normal versus DNA tumor virus-transformed cells. Hengstschlager, M., Knofler, m., Mullner, E. W., Ogris, E., Wintersberger, E., Wawra, E. J. Biol. Chem., 269: 13836-13842, 1994).”
The use of MAb to specifically target viral-infected cells is an approach which can leave normal or uninfected tissue or cells unharmed. MAb's may be used to construct therapeutic reagents with selectivity for certain populations of cells. Optionally, MAbs or other cell targeting proteins are linked to bioactive moieties to form biotherapeutic agents referred to as immunoconjugates, immunotoxins or fusion proteins, which can combine the selectivity of the targeting moiety with the potency of the bioactive moiety. Embodiments of the invention are directed to the use of anti-TK1 antibody to inhibit cell proliferation in cells that synthesize and express TK1 such as virally-infected cells as well as the use of anti-TK1 antibodies in diagnosis of viral infection.